Teaching molecular techniques at home: Molecular biology labs that can be performed anywhere and enable hands-on learning of restriction digestion/ligation and DNA amplification.

COVID 19 has changed about every aspect of life including how we teach in higher education. Laboratory experiments vital for learning hands-on techniques are limited due to social distancing requirements and increased numbers of distance-learning students. The solution to loss of hands-on activities has been to compensate with virtual laboratory modules. Although virtual labs are engaging and offer a simulated hands-on approach to teaching essential molecular techniques, these simulations do not replace hands-on experience. I designed two molecular biology laboratory exercises in response to the current teaching limitations that can be completed 'at-home' and enable low cost hands-on instruction of essential molecular techniques in any distance-learning environment including during the COVID 19 pandemic.

Unraveling the role of the enigmatic MatK maturase in chloroplast group IIA intron excision.

Maturases are prokaryotic enzymes that aid self-excision of introns in precursor RNAs and have evolutionary ties to the nuclear spliceosome. Both the mitochondria and chloroplast, due to their prokaryotic origin, encode a single intron maturase, MatR for the mitochondria and MatK for the chloroplast. MatK is proposed to aid excision of seven different chloroplast group IIA introns that reside within precursor RNAs for essential elements of chloroplast function. We have developed an in vitro activity assay to test chloroplast group IIA intron excision. Using this assay, we demonstrate self-excision of the group IIA intron of the second intron of rps12 and the group IIA intron of rpl2. We further show that the addition of heterologously expressed MatK protein increases efficiency of group IIA intron self-splicing for the second intron of rps12 but not the group IIA intron of rpl2. These data, to our knowledge, provide the first direct evidence of MatK's maturase activity.

Alternative translation initiation codons for the plastid maturase MatK: unraveling the pseudogene misconception in the Orchidaceae.

BACKGROUND: The plastid maturase MatK has been implicated as a possible model for the evolutionary "missing link" between prokaryotic and eukaryotic splicing machinery. This evolutionary implication has sparked investigations concerning the function of this unusual maturase. Intron targets of MatK activity suggest that this is an essential enzyme for plastid function. The matK gene, however, is described as a pseudogene in many photosynthetic orchid species due to presence of premature stop codons in translations, and its high rate of nucleotide and amino acid substitution. RESULTS: Sequence analysis of the matK gene from orchids identified an out-of-frame alternative AUG initiation codon upstream from the consensus initiation codon used for translation in other angiosperms. We demonstrate translation from the alternative initiation codon generates a conserved MatK reading frame. We confirm that MatK protein is expressed and functions in sample orchids currently described as having a matK pseudogene using immunodetection and reverse-transcription methods. We demonstrate using phylogenetic analysis that this alternative initiation codon emerged de novo within the Orchidaceae, with several reversal events at the basal lineage and deep in orchid history. CONCLUSION: These findings suggest a novel evolutionary shift for expression of matK in the Orchidaceae and support the function of MatK as a group II intron maturase in the plastid genome of land plants including the orchids.

Phospholipase D family interactions with the cytoskeleton: isoform delta promotes plasma membrane anchoring of cortical microtubules.

Phospholipase D (PLD) is a key enzyme in signal transduction - mediating plant responses to various environmental stresses including drought and salinity. Isotype PLDδ interacts with the microtubule cytoskeleton, although it is unclear if, or how, each of the 12 PLD isotypes in Arabidopsis may be involved mechanistically. We employed RNA interference in epidermal cells of Allium porrum L. (leek) leaves, in which the developmental reorientation of cortical microtubule arrays to a longitudinal direction is highly sensitive to experimental manipulation. Using particle bombardment and transient transformation with synthetic siRNAs targeting AtPLDα, ß, γ, δ, ॉ and ζ, we examined the effect of 'cross-target' silencing orthologous A. porrum genes on microtubule reorientation dynamics during cell elongation. Co-transformation of individual siRNAs together with a GFP-MBD microtubule-reporter gene revealed that siRNAs targeting AtPLDδ promoted, whereas siRNAs targeting AtPLDß and γ reduced, longitudinal microtubule orientation in A. porrum. These PLD isotypes, therefore, interact, directly or indirectly, with the cytoskeleton and the microtubule-plasma membrane interface. The unique response of PLDδ to silencing, along with its exclusive localisation to the plasma membrane, indicates that this isotype is specifically involved in promoting microtubule-membrane anchorage.

Dynamic changes in the mitochondrial electron transport chain underpinning cold acclimation of leaf respiration.

We examined the effect of short- and long-term changes in temperature on gene expression, protein abundance, and the activity of the alternative oxidase and cytochrome oxidase pathways (AOP and COP, respectively) in Arabidopsis thaliana. The AOP was more sensitive to short-term changes in temperature than the COP, with partitioning to the AOP decreasing significantly below a threshold temperature of 20 degrees C. AOP activity was increased in leaves, which had been shifted to the cold for several days, but this response was transient, with AOP activity subsiding (and COP activity increasing) following the development of leaves in the cold. The transient increase in AOP activity in 10-d cold-shifted leaves was not associated with an increase in alternative oxidase (AOX) protein or AOX1a transcript abundance. By contrast, the amount of uncoupling protein was significantly increased in cold-developed leaves. In conjunction with this, transcript levels of the uncoupling protein-encoding gene UCP1 and the external NAD(P)H dehydrogenase-encoding gene NDB2 exhibited sustained increases following growth in the cold. The data suggest a role for each of these alternative non-phosphorylating bypasses of mitochondrial electron transport at different points in time following exposure to cold, with increased AOP activity being important only in the early stages of cold treatment.

Evaluating evolutionary constraint on the rapidly evolving gene matK using protein composition.

The rapidly evolving chloroplast matK gene has nucleotide and amino acid substitution rates suggestive of progression toward a pseudogene state. However, molecular evidence has demonstrated that matK is expressed and functional. We explore in this paper the underlying factors behind the mode and tempo of matK evolution that allow this protein coding gene to accommodate such elevated rates of substitution and yet maintain functionality. Conservative amino acid replacement may reconcile the fast evolutionary rate in matK with conservation in protein function. Based on this premise, we have examined putative amino acid sequences for MATK from across green plants to determine constraint on this protein as indicated by variation in composition of amino acid side chain category. Amino acids in the MATK ORF were divided into six categories based on chemical properties of their side chains: nonpolar, uncharged (pH 7), basic, acidic, aromatic, and "special" (amino acids that specifically affect protein structure, i.e., proline, glycine, and cysteine). The amount of standard deviation (SD) in side chain composition was used as a measure of variation and constraint, where a low SD implied high evolutionary constraint and a high SD implied low constraint. Further, we used secondary structure prediction to evaluate if conservation observed in side chain composition was reflected in stable predicted structure. The results of this study demonstrate evolutionary constraint on MATK, identify three regions of functional importance, show highly conserved secondary structure, and support the putative function of MATK as a group II intron maturase.

Expression of matK: functional and evolutionary implications.

Strong phylogenetic signal from matK has rendered it an invaluable gene in plant systematic and evolutionary studies at various evolutionary depths. Further, matK is proposed as the only chloroplast-encoded group II intron maturase, thus implicating MATK in chloroplast posttranscriptional processing. For a protein-coding gene, matK has an unusual evolutionary mode and tempo, including relatively high substitution rates at both the nucleotide and amino acids levels. These evolutionary features have raised questions about matK function. In the current study, we examined matK RNA and protein from representative land plant species to provide insight into functional aspects of this unusual gene. We report the first evidence of a transcript for matK separate from the trnK precursor and demonstrate that a full-length MATK protein exists in five angiosperm species. We also show that matK RNA and protein levels are regulated by light and developmental stage, suggesting functional roles for this putative maturase. Specifically, matK expression increased after etiolation and decreased at 4 weeks after germination. This work provides evidence for the expression of the only putative chloroplast-encoded group II intron maturase and insight into regulation mechanisms relating to plant development and, indirectly, to photosynthesis.